Apparatus and methods relating to high speed raman spectroscopy

ABSTRACT

Systems and methods for rapid Raman spectroscopy. The speed is improved by providing light from a sample to a light-dispersive element, such as a holographic grating, in a pattern that inversely complements distortion caused by the grating. For example, if the grating imparts a curve to the spectral lines emanating from the grating, then the light is inserted into the grating in a curve in the opposite direction. Also calibration light guides able to transmit a known, or standard, light to the detection or spectroscopy system. The calibration light guide can be useful both with traditional light transmission guides and with the light transmission guides of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from U.S. provisionalpatent application No. 60/154,070, filed Sep. 14, 1999.

BACKGROUND OF THE INVENTION

[0002] When monochromatic light such as laser light strikes a sample,almost all of the light is scattered elastically, which is calledRayleigh scattering. This Rayleigh scattered light undergoes no changein energy or frequency. However, a very small portion of the light, ˜1in 10⁸, is scattered inelastically, which is called Raman scattering.This light does undergo a change in energy and frequency, and the changecorresponds to an excitation of the illuminated molecular system, mostoften excitation of vibrational modes. Measuring the intensity of theRaman scattered photons as a function of the frequency differenceprovides a Raman spectrum. Raman peaks are typically narrow (a fewwavenumbers) and in many cases can be associated with the vibration of aspecific chemical bond (or normal mode dominated by the vibration of asingle functional group) in a molecule. As such, it is a “fingerprint”for the presence of various molecular species and can be used for bothqualitative identification and quantitative determination. AnalyticalRaman Spectroscopy, Chemical Analysis Series Vol. 114, J. G. Grasselliand B. J. Bulkin, Eds. (John Wiley, New York, 1991).

[0003] Raman spectroscopy has a variety of potential uses in vivo. Forexample, Raman spectra have been observed from various biologicaltissues including skin. Ozaki, Y.: “Medical application of Ramanspectroscopy” Appl. Spectr. Rev. 24:259-312, 1988; Manoharan, R., et al.“Histochemical analysis of biological tissues using Raman spectroscopy,”Spectro. Acta Part A, 52:215-249, 1996; Mahadevan-Jansen, A. andRichards-Kortum, R.: “Raman spectroscopy for the detection of cancersand precancers,” J. of Biomed. Op., 1:31-70, 1996. Identified Ramanscatterers in tissues include elastin, collagen, blood, lipid,tryptophan, tyrosine, carotenoid, myoglobin. Id. Most of the data hasbeen obtained from ex vivo tissue samples using Fourier-Transform (FT)Raman spectrometers. These data have demonstrated that Ramanspectroscopy has potential for diagnosis of diseases. Raman spectroscopymight also be used to monitor cutaneous drug delivery andpharmacokinetics during skin disease treatment. Schallreuter, K. U.:“Successful treatment of oxidative stress in vitiligo,” Skin Pharmacol.Appl. Skin Physiol. 12(3):132-8, 1999; Lawson, E. E., et al.“Interaction of salicylic acid with verrucae assessed by FT-Ramanspectroscopy,” J. Drug Target 5(5):343-51, 1998; Schallreuter, K. U., etal. “In vivo evidence for compromised phenylalanine metabolism invitiligo,” Biochem. Biophys. Res. Commun. 13; 243(2):395-9, 1998;Schallreuter, K. U., et al. “Oxybenzone oxidation following solarirradiation of skin: photoprotection versus antioxidant inactivation,”J. Invest. Dermatol. 106(3):583-6, 1996. Raman spectroscopy could alsopotentially be used to detect the presence of prohibited drugs used byathletes or specific drugs in drug abusers.

[0004] In order to enhance such uses, Raman measurements should be ableto be performed in vivo and quickly, preferably within seconds orsub-seconds. FT-Raman systems typically require as much as half an hourto acquire a spectrum and are typically bulky and not portable.Manoharan, R., et al. “Histochemical analysis of biological tissuesusing Raman spectroscopy,” Spectro. Acta Part A, 52:215-249, 1996.Recently developed dispersive-type Raman systems based on fiber opticlight delivery and collection, compact diode lasers, and high efficiencyspectrograph-detector combinations, may be able to acquire in vivotissue Raman spectrum in seconds. Baraga, J.J., et al. “RapidNear-infrared Raman spectroscopy of human tissue with a spectrograph andCCD detector,” Appl. Spectro. 46:187-90, 1992; Kramer, J. R., et al.“Spectral diagnosis of human coronary artery: a clinical system for realtime analysis,” SPIE Proc. 2395:376-82, 1995; Mahadevan-Jansen, A., etal. “Development of a fiber optic probe to measure NIR Raman spectra ofcervical tissue in vivo,” Photochem. Photobiol. 68(3):427-31, 1998.

[0005] Thus, there is a need for Raman spectroscopy systems capable offast speeds or high quality results. The present invention providesthese and other advantages.

SUMMARY OF THE INVENTION

[0006] The present invention provides systems and methods for rapidRaman spectroscopy. The speed is improved by providing light from asample to a light-dispersive element, such as a holographic grating, ina pattern that inversely complements distortion caused by the grating orother device. For example, if the grating imparts a curve to thespectral lines emanating from the grating, then the light is insertedinto the grating in a curve in the opposite direction. These and otherfeatures of the present invention enhance the signal to noise ratio andimprove the spectral resolution of the system. The present inventionalso provides a calibration light guide able to transmit a known, orstandard, light to the detection or spectroscopy system. The calibrationlight guide can be useful both with traditional light transmissionguides and with the light transmission guides of the present invention.

[0007] In one aspect, the present invention provides a lighttransmission bundle suitable for use for Raman spectroscopy, the lighttransmission bundle comprising a proximal end and a distal end andcomprising at least 5 light guides, wherein the light guides arearranged in a substantially filled-in geometrical shape at the proximalend of the light transmission bundle and a substantially linear curve atthe distal end. In some embodiments, the substantially linear curve is aparabolic curve, and can be substantially identical to a curve of asubstantially linear line of light after it has been passed through aholographic grating, for example a volume phase technology (VPT)holographic grating. In some embodiments, the filled-in geometricalshape is a circle, although other shapes are also possible. (Unlessexpressly stated otherwise or clear from the context, all embodiments ofthe present invention can be mixed and matched.) The present inventionalso provides a calibration light guide, which can be one of the lightguides at the distal end of the light transmission bundle describedabove, for example at the center of the substantially linear curve.Typically, the proximal end of the calibration light guide is opticallyconnected to a calibration light source.

[0008] In another aspect, the present invention provides a Ramanspectrometer system comprising a detection light guide able to detectlight emanating from a sample. A distal end of the detection light guideis optically connected to a plane grating that is in turn opticallyconnected to a pixelated light detector operably connected to acontroller containing computer-implemented programming that detectslight impinging on detection pixels in the pixelated light detector. Alight transmissive portion of the distal end of the detection lightguide is arranged in a substantially inverse shape that is complementaryto a distortion to the light caused by passing the light through theplane grating, to provide light in a substantially straight line at thepixelated light detector. In some embodiments, the light guide issimilar to that described above.

[0009] The Raman spectrometer system can further comprise amonochromatic illumination light source that provides illumination lightto the sample, and an illumination light guide and a probe located atthe distal end of the illumination light guide, wherein the illuminationlight guide transmits the monochromatic illumination light from thelight source to the probe, which in turn transmits the illuminationlight to the sample. The monochromatic illumination light source can bean infrared laser, for example having a power of at least about 250 mW,a wavelength of about 785 nm, and a power of about 300 mW. Theillumination light guide can be a single optical fiber having a diameterless than about 200 μm. Preferably, the proximal end of the detectionlight guide is optically connected to the probe.

[0010] The probe can further comprise a compound parabolic concentrator(CPC) optically connected at the proximal end of the detection lightguide, wherein the compound parabolic concentrator collects lightemanating from the sample and concentrates it and transmits it into thedistal end of detection light guide. Alternatively, the proximal end ofthe detection light bundle can have a large a diameter, for examplegreater than about 1 mm, and then the detection probe may not comprise aCPC. In some embodiments, the probe is sized to provide an illuminationspot on the sample that is substantially larger than a detection spotdetected by the probe.

[0011] The Raman spectrometer system can further comprise a calibrationlight guide optically connected to a calibration light source. Also, thedetector preferably comprises an array of detection pixels and thedetection light guide preferably comprises a bundle comprising enoughlight guides to substantially fill a column or row of the array, whereinthe bundle comprises more than about 50 light guides. The light guidescan be selected from the group consisting of an optical fiber, a liquidlight guide and a hollow reflective light guide. Also, the system ispreferably portable.

[0012] The present invention further provides means for and steps ofachieving the various aspects, embodiments and features of suchinvention.

[0013] In a further aspect, the present invention provides methods ofmaking a light transmission bundle comprising a) providing at least 5light guides, b) arranging the light guides in a filled-in geometricalshape at a proximal end of the light transmission bundle, and c)arranging the light guides in a substantially linear curve at the distalend of the light transmission bundle. In some embodiments, the methodsfurther comprise adding a calibration light guide to the lighttransmission bundle.

[0014] In another aspect, the present invention provides methods oftaking a Raman measurement comprising a) illuminating a sample underconditions and for a time sufficient to induce measurable Ramanscattered light to emanate from the sample, b) collecting the emanatinglight, c) providing the emanating light with a substantially inverseshape that is complementary to a distortion to the light caused bypassing the light through a light-dispersive element, d) passing thelight having the substantially inverse shape through the plane gratingto provide substantially straight spectral lines, and e) performingRaman spectroscopic analysis on the substantially straight spectrallines. In some embodiments, providing the substantially inverse shapefurther comprises transmitting the emanating light through a detectionlight guide wherein the light transmissive portion of the distal end ofthe detection light guide is arranged in a substantially inverse shapethat is complementary to a distortion to the light caused by passing thelight through the light transmissive element, and wherein the lighttransmissive element comprises a plane grating. Illuminating the sampleand collecting the emanating light can be done through a probe opticallyconnected to an illumination light source and to the detection lightguide, for example by providing an illumination spot on the sample thatis substantially larger than the detection spot detected by the probe.The methods can further comprise providing calibration light, and can beperformed using a portable Raman system, and in less than about 1second.

[0015] These and other aspects of the present invention will becomeevident upon reference to the following Detailed Description andattached drawings. In addition, various references are set forth hereinthat describe in more detail certain apparatus and methods; all suchreferences are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 provides an image of light from a 100-μm slit passedthrough a holographic plane grating, demonstrating image aberration.

[0017]FIG. 2 depicts a graph of the horizontal displacement of aspectral line as in FIG. 1, with the displacement rounded to 27-μmpixels.

[0018]FIG. 3 provides a schematic block diagram of a rapid Ramanspectrometer system.

[0019]FIG. 4 provides a schematic block diagram of a rapid Ramanmicrospectrophotometer system.

[0020]FIG. 5 provides a schematic block diagram of a probe for Ramanmeasurement in vivo or for other distant samples, the Figure alsodepicting the proximal and distal ends of a detection light guideaccording to the present invention.

[0021]FIG. 6 provides an image of spectral lines from light that hasbeen supplied to the HoloSpec spectrograph in a curve comprising 58optical fibers that is inversely complementary to aberration depicted inFIG. 1. The dark spots in the middle of the spectral lines are from thecalibration fiber, which was not illuminated in the image.

[0022]FIG. 7 provides an in vivo Raman spectrum from the palm of ahealthy volunteer obtained with an integration time of 5 seconds usingthe Raman system of the present invention.

[0023]FIG. 8 provides an in vivo Raman spectrum from the back of thehand of a healthy volunteer with an integration time of 5 seconds usingthe Raman system of the present invention.

[0024]FIG. 9 provides a Raman spectrum of BaSO₄ coating on glass slideobtained with an integration time of 1 seconds using the Raman system ofthe present invention.

[0025]FIG. 10 provides a Raman spectrum obtained directly from anacetominophen (Tylenol®) pill obtained with an integration time of 0.5seconds using the Raman system of the present invention.

[0026]FIG. 11 provides a Raman spectrum obtained directly from anaspirin pill obtained with an integration time of 0.5 seconds using theRaman system of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides high speed Raman systems forspectroscopy, such as in vivo spectroscopy, and other spectral analyses.The performance of the Raman systems is improved by providing a lightcollection or detection device that displays light emanating from asample into a holographic grating, or other device that separates thelight into its spectral components, in a pattern that inverselycomplements any distortion caused by the grating or other device. Forexample, if the grating imparts a curve to the spectral lines emanatingfrom the grating, then the light collection device corrects thedistortion by inserting the light into the grating in a curve in theopposite direction. These and other features of the present inventionenhance the signal to noise ratio and improve the spectral resolution ofthe system. The present invention also provides a calibration lightguide able to transmit a known, or standard, light to the detection orspectroscopy system. The calibration light guide can be useful both withtraditional light transmission guides and with the light transmissionguides of the present invention.

[0028] One of the difficulties with previous high speed Raman systemsthat use a plane grating, and other Raman or other optical systems thattransmit light through a plane grating, is that the light is distortedby the plane grating. For example, a straight line of light (such aslight passed through a slit) typically comes out of the grating assubstantially linear curve, typically a parabolic curve. James, J. F.,and Sternburg, R. S.: “The design of optical spectrometers,” Chapman andHall Ltd., London, England, 1969. This phenomenon can be due to the factthat the light rays from different positions along the length of theslit are incident on the grating with varying amounts of obliqueness.For spectrographs having a short focal length, such as a holographicspectrograph such as the Kaiser HoloSpec spectrograph (Kaiser OpticalSystems. Inc., Ann Arbor, Mich.), the obliqueness causes a significantdistortion that can affect the performance of a detector downstream inthe light path. As an example of this, FIG. 1 shows the spectral linesresulting from passing light from a mercury-argon calibration lampthrough a 100 μm slit and then through a Kaiser HoloSpec spectrograph.The curvature of the spectral lines is apparent in FIG. 1. FIG. 2 showsin graphical form the horizontal displacement of a spectral line fromFIG. 1 in graphic form, with the displacement rounded to 27-μm pixels.The maximum horizontal displacement in FIG. 2 is 5 pixels.

[0029] One of the well-known approaches for improving the performance ofa spectrometer is to combine the signal from an entire spectral line foranalysis. This can be called “hardware binning,” and in a charge-coupleddevice (CCD) the binning can be performed before the signal is read outby the preamplifier. For signal levels that are limited by readoutnoise, such as weak Raman signal measurement, hardware binning improvessignal to noise ratio (SIN) linearly with the number of pixels groupedtogether. The image aberration described in the previous paragraphdetracts from the effectiveness of hardware binning because the entirespectral line no longer hits a single line (row or column) of pixels inthe detector. This can decrease the spectral resolution and the S/N. Itcan also cause problems with wavelength calibration. Binning can also bedone by software after the signal is read out. However, such softwarebinning typically improves the S/N only by as much as the square root ofthe number of pixels added together. Hence, hardware binning ispreferred for maximum S/N. Other approaches bin the 11 segments shown inFIG. 2 using hardware binning and then shifting the appropriate numberof pixels before summing them together using software binning. KaiserOptical Systems, Inc. “HoloSpec VPT System Operations Manual,” 1994.This may be termed a “combined hardware and software binning procedure.”The previous approach of acquiring the whole image first and then addingall the pixels along the curved line together by software binning can betermed a “complete software binning procedure.”

[0030] One of the features of the present invention corrects the imageaberration and allows hardware binning of a whole spectral line. Asshown in FIG. 5 and described more fully below, the output from thedetection light guide is provided to the grating in a shape that issubstantially inversely proportional to the aberration caused by thegrating. For example, where the grating imparts a parabolic curve to thelight, the output of the detection light guide forms an inverse orcomplementary curve, as shown in FIG. 5, so the light emanating from thegrating onto the detector is a straight line, as shown in FIG. 6. Thisallows full hardware binning of the entire vertical line without losingresolution and without reducing S/N. Compared to the combined hardwareand software binning procedure, the S/N improvement can be up to atleast about 11^(1/2) or 3.3 times or more, and compared to the completesoftware binning procedure, the S/N improvement can be up to at leastabout 256^(1/2) or 16 times.or more.

[0031] Definitions

[0032] The following paragraphs provide definitions of some of the termsused herein. All terms used herein, including those specificallydescribed below in this section, are used in accordance with theirordinary meanings unless the context or definition indicates otherwise.Also unless indicated otherwise, except within the claims the use of“or” includes “and” and vice-versa. Non-limiting terms are not to beconstrued as limiting unless expressly stated (for example, “including”means “including without limitation” unless expressly stated otherwise).

[0033] An “illumination light path” is a light path from a light sourceto a sample, while a “detection light path” is a light path for lightemanating a sample to a detector. Light emanating from a sample includeslight that reflects from a sample, is transmitted through a sample, oris created within the sample, for example, fluorescent light that iscreated within a sample pursuant to excitation with an appropriatewavelength of light (typically UV or blue light). In the presentapplication, such emanating comprises light that has been Ramanscattered.

[0034] A “controller” is a device that is capable of controlling adetector or other elements of the apparatus and methods of the presentinvention. For example, the controller may control the pixels of apixelated light detector or compile or interpret data obtained from thedetector. Typically, a controller is a computer or other devicecomprising a central processing unit (CPU). Controllers are well knownin the art and selection of a desirable controller for a particularaspect of the present invention is within the scope of the art in viewof the present disclosure.

[0035] “Upstream” and “downstream” are used in their traditional sensewherein upstream indicates closer to a light source, while downstreamindicates farther away from a light source. Similarly, “proximal”indicates upstream and “distal” indicates downstream.

[0036] The terms set forth in this application are not to be interpretedin the claims as indicating a “means plus function” relationship unlessthe word “means” is specifically recited in the claims. This is alsotrue of the term “step” for process or method claims.

[0037] Other terms and phrases in this application are defined inaccordance with the above definitions, and in other portions of thisapplication.

[0038] The Figures

[0039] Turning to the figures, as noted above FIGS. 1 and 2 depictcurved spectral lines from a plane grating. FIG. 3 depicts a schematicblock diagram of a rapid Raman spectrometer system according to oneembodiment of the invention. In the figure, a light source 4, preferablya monochromatic light source such as laser, for example an infraredlaser such as an external cavity stabilized diode laser (785 nm, 300 mW,SDL Inc.), provides light capable of inducing Raman scattering in asample, such as tissue. Preferably, the light source minimizesinterference from non-desired sources. For example, if the sample is atissue, then the light source can be selected to minimize fluorescencefrom the sample and the absorption of photons emanating from melanin.The light source 4 is optically coupled to the proximal end of anillumination light transmission guide 6. In FIG. 3, the illuminationlight transmission guide 6 is a 100-μm optic fiber. The illuminationlight transmission guide 6, and other light guides of the presentinvention, can also be a liquid light guide or hollow reflective lightguide or lens system, or a bundle of any of such light guides or otherlight guides able to transmit the illumination or detection light, orother light, as desired.

[0040] The illumination light transmission guide 6 is opticallyconnected at its distal end to a probe 8, for example via a SMAconnector. The probe 8 transmits thevn illumination light to the sample9, and then gathers the return light emanating from the sample, whichreturn light comprises Raman photons. The light collected by probe 8 istransmitted to a spectrograph 2, such as a holographic transmissivespectrograph (Kaiser, HoloSpec-f/2.2-NIR), through a detection lightguide 10. As noted above, the detection light guide that transmits thedetection light can be an optical fiber, a liquid light guide or hollowreflective light guide or lens system, or a bundle of any of such lightguides or other light guides able to transmit the light. In a preferredembodiment, the detection light guide comprises a bundle of light guidesbecause it is relatively simple to control the geometry of the lightguides in the bundle so that the bundle can have one shape at theproximal end of the detection light guide (i.e., the portion of thedetection light guide 10 at the probe) and a second shape at the distalend of the light guide (i.e., the portion of the detection light guide10 at the spectrograph). A calibration light guide 12 provides lightfrom a calibration light source to the spectrograph 2.

[0041] The light from the detection light guide 10 is fed through a lens14 and into a plane grating 16. In preferred embodiments the planegrating is a holographic grating, such as a volume phase technology(VPT) holographic grating (Kaiser, HSG-785-LF). The plane grating 16disperses the incoming light through a second lens 18 onto the detector20. The detector can be pixelated, such as a charge coupled device(CCD), such as an LN/CCD-1024EHRB from Princeton Instruments, chargeinjection device (CID), intensified CCD detector, photomultiplier tube(PMT) detector array, photo-diode array (PDA), intensified PDA and anavalanche photo-diode (APD) array. In a preferred embodiment, thedetector is a back-illuminated, deep depletion CCD detector withenhanced QE and reduced etaloning in the NIR (QE≧70% at 900 nm), isliquid nitrogen cooled, and its S/N is read-out noise limited whenacquiring weak Raman signals. The detector in one example has 1024×256pixels (27-μm×27-μm) in a 27.6-mm×6.9-mm size rectangular array andallows vertical binning (Princeton Instruments, LN/CCD-1024EHRB). Thedetector 20 is operably connected to and controlled by a controller 22,which can be a PC computer. The Raman spectra are preferably displayedon the computer screen in real time and can also be saved for futuredisplay or further analysis.

[0042] In one embodiment, the plane grating 16 covers the low frequency“fingerprint” Raman bands from 0 up to 2080 cm⁻¹. In another embodiment,the spectrograph 2 has an f-number, such as 2.2, that matches thenumerical aperture (NA) of the fiber bundle (e.g., NA=0.22). Couplingoptics in the spectrograph preferably provide 1:1 imaging with minimaloptical losses due to NA matching between the spectrograph and thefibers. This spectrograph has 5 times better throughput and betterspectral resolution compared to a traditional f/4, ¼-meter imagingCzerny-Turner spectrograph used in conventional dispersive Raman systems(see, e.g., Owen, H., et al., “New spectroscopic instrument based onvolume holographic optical elements.” SPIE Proceed., 2406, 1995).

[0043] The whole spectrometer system of this and other embodiments ofthe present invention can be permanent or can be portable, for exampleit can be movable on a cart for outpatient clinical data acquisition.

[0044]FIG. 4 depicts an example of a rapid Raman microspectrophotometersystem according to the present invention. The system provides bothimaging and spectral analysis. The system comprises up to four lightsources, monochromatic (here, a laser) light source 4, calibration lightsource 13, alignment light source 19 and halogen lamp 36. Calibrationlight source 13 provides alignment light that is transmitted into thesystem. Alignment light source 19 provides light that travels up to themicroscope to identify the specific spot being examined in the sample.Light from the monochromatic light source 4 or halogen lamp 36 istransmitted to a sample 9, via one or more mirrors 24, condenser lenses26 or illumination light guides 6. Light emanating from the sample istransmitted to eyepiece 32, imaging device 38 such as a CCD camera, orRaman spectrometer 40. One or more of the devices, in the figure theRaman spectrograph 2 and imaging device 38, can be operably connected toa controller 22. The system can include a band pass filter 34 betweenthe illumination light source and the sample to eliminate Raman spectraand fluorescence from the illumination light path, a notch filter 30between the sample and the spectrograph 2 to filter out illuminationlight from monochromatic light source 4, and an optional narrow bandpass filter 35 in front of the imaging device to select Raman bands ofinterest for imaging. Other filters, mirrors, lenses and other opticalcomponents can be selected by a person of ordinary skill in the art inview of the present specification.

[0045]FIG. 5 depicts a probe for taking Raman measurements, and theproximal and distal ends of a detection light guide according to thepresent invention. In the probe 8, a collimator lens 42 collimates theillumination light coming out of illumination light guide 6, and a bandpass filter 34 filters out the Raman signals and the autofluorescencesignals that are generated by passing the illumination light through theillumination light guide 6. Mirror 24 deflects the illumination lightbeam to the sample 9. In an alternative embodiment, the illuminationlight passes through a holographic band pass filter (Kaiser OpticalSystems, Inc.) that turns the illumination light beam 90°. The beam isthen deflected to the sample by a mirror. A pair of lenses 15, 17, forexample, two one-inch diameter f/2 quartz lenses, collects the scatteredlight from the sample surface. The collimator lens 15 is preferablypositioned so that its focal point is located at the sample surface,providing a collimated beam between the two lenses. A notch filter 30,such as a holographic notch plus filter (Kaiser), can be placed betweenthe collimator lens 15, and the focusing lens 17, to block the Rayleighscattered light and pass the frequency-shifted Raman signal. Focusinglens 17 focuses the filtered beam onto the proximal end of detectionlight guide 10. Detection light guide 10 then transmits the light to theRaman spectrometer.

[0046] In one embodiment, detection light guide 10 comprises 100 μmoptical fibers 44 packed into a first shape, such as a filled-ingeometric shape such as a circle (as in the Figure), a square, arectangle or a hexagon, at the proximal, or input, end 46. The shape canbe other than filled-in if desired. The fibers are arranged in a secondshape, such as curved line, at the distal, or output, end 48. Inparticular, the optical fibers 44 or other light transmissive portion ofthe detection light guide 10 are arranged such that they provide asubstantially inverse shape to the distortion to the light caused bypassing the light through the plane grating, which means that the shapeis complementary to such distortion so that the light emanating from theplane grating 16 is in a straight line (or other shape if so desired).For example, in one embodiment the shape at the distal end 48 ofdetection light guide 10 is a parabolic curve that has substantially thesame horizontal displacement as shown in FIGS. 1 and 2 but in thereverse direction. A calibration fiber 50 can be added to the distal end48 of detection light guide 10. Again referring to the curve in FIGS. 1and 2 for exemplary purposes, a parabolic line or curve can be fitted tothe graph in FIG. 2 by linear regression line fitting or can beapproximated by five segments of straight lines.

[0047]FIG. 6 shows a CCD image of light from a mercury-argon calibrationlamp. The light was carried by a detection light guide 10 comprising abundle of about 58 100 μm light guides arranged in an inverse curve asdiscussed above and then passed through a plane grating to give spectrallines. The spectral lines of the light are substantially straight. Thedark spots in the center of the spectral lines are from a 50-μmcalibration light guide disposed at the center of the bundle that wasnot illuminated. Accordingly, the light can be hardware binned withoutlosing resolution and without reducing S/N.

[0048] Preferably, the detection light guide 10 is a bundle andcomprises as many fibers as will fit the vertical or horizontal (orother straight-line characteristic) size of the detector. For example,for the image in FIG. 6, the CCD had a height of about 6.9 mm, and about58×100-μm optic fibers and 1×50 μm optic fiber were fitted into thedetection light guide 10.

[0049] At the proximal end of the detection light guide 10 the fiberswere packed into a 1.6 mm diameter area. Therefore the measurement ordetection spot size on the sample surface was also 1.6 mm. If desired, acompound parabolic concentrator (CPC) can be used to collect more lightfrom the sample. However, the proximal end diameter of the fiber bundle,which is restricted by the vertical size of the CCD array and thedesired spectral resolution, may not be as big as the output aperture ofthe CPC to fully make use of the light collection capability of the CPC.Also the laser beam may need to be focused smaller than the inputaperture diameter of the CPC, therefore, restricting the totalillumination power to prevent tissue damage. Accordingly, in anotherembodiment it is preferred not to use a CPC, but collect light directlyfrom a large spot on the sample. Preferably, the illumination spot islarger than the detection spot to increase the total allowableillumination power. The Raman signal generated by the large beam maydiffuse into the measurement spot and be collected.

[0050] In other aspects, the present invention provides methods ofmaking and using the devices and systems described herein. For example,the present invention provides methods of making a light transmissionguide or bundle as described herein, which methods can include providingat least 5 light guides, arranging the light guides in a substantiallyfilled-in geometrical shape at a proximal end of the light transmissionbundle, and arranging the light guides in a substantially linear curveat the distal end of the light transmission bundle. The order of thesteps in these methods is not critical. In some embodiments, the methodscan further include arranging the light transmissive portion or elementof the light guide in a substantially parabolic curve at the distal endof the light transmission guide, the parabolic curve being substantiallyidentical to a curve of a substantially linear line of light after ithas been passed through a holographic grating if desired. Similarly, thefilled-in geometrical shape can be a circle or other desired shape. Inother embodiments, the methods comprise providing a calibration lightguide to the light transmission bundle. The calibration light guide canbe useful both with traditional light transmission guides and with thelight transmission guides of the present invention.

[0051] In further aspects, the present invention provides methods oftaking a Raman measurement comprising illuminating a sample underconditions and for a time sufficient to induce measurable Ramanscattered light to emanate from the sample, collecting the emanatinglight, transmitting the emanating light through a detection light guideas described herein, passing the light through the plane grating toprovide substantially straight spectral lines, and performing Ramanspectroscopic analysis, such as display, measurement, comparison orother desired functions on the substantially straight spectral lines. Inother aspects, the methods comprise providing the light in a particularshape that is complementary to the shape imparted by thelight-dispersive element (such as the plane grating) via the use ofcurved mirrors or other optical elements able to control the shape ofthe light. The present invention also includes systems comprising suchalternative approaches to providing complementary-shaped light.

[0052] In some embodiments, the methods further comprise illuminating adistant sample, such as an in vivo sample or a sample in a difficult toreach location in a machine, and collecting the emanating light from thesample through a probe such as those as described herein. The methodscan also be performed using a portable Raman system. For example, thesystem can be carried on a cart or in a carry-bag so that it can movefrom one person or machine to another. The Raman measurements can beobtained in relatively short times, typically less than about 5 seconds,generally less than about 2 seconds, preferably less than about 1second, and further preferably less than about 0.5 seconds

EXAMPLES

[0053] A Raman spectroscopy system was built with a detection lightguide comprising a bundle of 58×100 μm and 1×50 μm optic fiber lightguides arranged in an inverse curve that can be described by thecoordinates x=0.000011904y²+0.000194553y−4.4485278. At the proximal endof the detection light guide the fibers were packed into a 1.6 mmdiameter area. Therefore the measurement or detection spot size on thesample surface was also 1.6 mm. The system also had a volume phasetechnology (VPT) holographic grating (Kaiser, HSG-785-LF) and anLN/CCD-1024EHRB charge coupled device (CCD) (Princeton Instruments). Thespectral lines of the light emanating from the sample were thussubstantially straight at the CCD and hardware binning of the spectrallines was performed.

[0054]FIG. 7 shows an in vivo Raman spectrum from the palm of a healthyvolunteer obtained with an integration time of 5 seconds using thesystem. The major Raman peaks shown in the graph are consistent withspectra published in literature. Caspers, P. J., et al., “In vitro andin vivo Raman spectroscopy of human skin,” Biospectro., 4:S31-S39, 1998;Gniadecka, M., et al., “Distinctive molecular abnormalities in benignand malignant skin lesions: Studies by Raman spectroscopy,” Photochem.Photobiol., 66:418-423, 1997; Gniadecka, M., et al., “Diagnosis of basalcell carcinoma by Raman spectroscopy,” J. Raman Spectrosc., 28:125-130,1997; Williams, A. C., et al., “Fourier transform Raman spectroscopy: Anovel application for examining human stratum corneum,” Int. J. Pharm.,81, R11-14, 1992; Barry, B. W., et al., “Fourier transform Raman andinfrared vibrational study of human skin: Assignment of spectral bands,”J. of Raman Spectro., 23:641-5, 1992; Edwards, H. G. M., et al., “Novelspectroscopic deconvolution procedure for complex biological systems:Vibrational components in the FT-Raman spectra of ice-man andcontemporary skin,” J. Chem. Soc. Faraday Trans., 91:3883-7, 1995;Williams, A. C., et al., “A critical comparison of some Ramanspectroscopic techniques for studies of human stratum corneum,” PharmaRes., 10:1642-1647, 1993; Williams, A. C., et al., “Comparison ofFourier transform Raman spectra of mammalian and reptilian skin,” Anal.,119:563-566, 1994; Williams, A. C., et al., “Raman spectra of humankeratotic biopolymers: Skin, callus, hair and nail,” J. RamanSpectrosc., 25: 95-98, 1994; Barry, B. W., et al., “Fourier transformRaman and IR spectra of snake skin,” Spectrochim. Acta, Part A,49:801-807, 1993; Edwards H. G. M., et al., “Raman spectroscopic studiesof the skin of the Sahara sand viper, the carpet python and the Americanblack rat snake,” Spectrochim. Acta. Part A, 49:913-919, 1993.

[0055]FIG. 8 shows an in vivo Raman spectrum from the hand dorsum of ahealthy volunteer with an integration time of 5 seconds. The major Ramanpeaks shown in the graph are consistent with spectra published inliterature.

[0056]FIG. 9 shows a Raman spectrum of a BaSO₄ coating on a glass slideusing an integration time of 1 second.

[0057]FIG. 10 provides a Raman spectrum obtained directly from a Tylenolpill. No sample preparations were performed prior to obtaining thespectrum. The integration time was 0.5 seconds.

[0058]FIG. 11 provides a Raman spectrum obtained directly from anAspirin pill. No sample preparations were performed prior to obtainingthe spectrum. The integration time was 0.5 seconds.

[0059] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

What is claimed is:
 1. A light transmission bundle suitable for use forRaman spectroscopy, the light transmission bundle comprising a proximalend and a distal end and comprising at least 5 light guides, wherein thelight guides are arranged in a substantially filled-in geometrical shapeat the proximal end of the light transmission bundle and a substantiallylinear curve at the distal end.
 2. The light transmission bundle ofclaim 1 wherein the substantially linear curve is a parabolic curve. 3.The light transmission bundle of claim 2 wherein the parabolic curve issubstantially identical to a curve of a substantially linear line oflight after it has been passed through a holographic grating.
 4. Thelight transmission bundle of claim 3 wherein the holographic grating isa volume phase technology (VPT) holographic grating.
 5. The lighttransmission bundle of claim 1 wherein the filled-in geometrical shapeis a circle.
 6. The light transmission bundle of claim 1 wherein atleast one of the light guides at the distal end of the lighttransmission bundle is a calibration light guide.
 7. The lighttransmission bundle of claim 6 the calibration light guide is disposedat the center of the substantially linear curve.
 8. The lighttransmission bundle of claim 6 or 7 wherein a proximal end of thecalibration light guide is optically connected to a calibration lightsource.
 9. A Raman spectrometer system comprising a detection lightguide able to detect light emanating from a sample, a distal end of thedetection light guide optically connected to a plane grating that is inturn optically connected to a pixelated light detector operablyconnected to a controller containing computer-implemented programmingthat detects light impinging on detection pixels in the pixelated lightdetector, wherein a light transmissive portion of the distal end of thedetection light guide is arranged in a substantially inverse shape thatis complementary to a distortion to the light caused by passing thelight through the plane grating, to provide light in a substantiallystraight line at the pixelated light detector.
 10. The Ramanspectrometer system of claim 9 wherein the detection light guidecomprises a light transmission bundle comprising at least 5 lightguides, wherein the light guides are arranged in a filled-in geometricalshape at a proximal end of the light transmission bundle and asubstantially linear curve at the distal end, and the plane gratingcomprises a holographic grating.
 11. The Raman spectrometer system ofclaim 10 wherein the system further comprises a monochromaticillumination light source that provides illumination light to thesample.
 12. The Raman spectrometer system of claim 11 wherein the systemfurther comprises an illumination light guide and a probe located at thedistal end of the illumination light guide, wherein the illuminationlight guide transmits the monochromatic illumination light from thelight source to the probe, which in turn transmits the illuminationlight to the sample.
 13. The Raman spectrometer system of claim 11wherein the monochromatic illumination light source comprises aninfrared laser.
 14. The Raman spectrometer system of claim 12 whereinthe illumination light guide comprises a single optical fiber having adiameter less than about 200 μm.
 15. The Raman spectrometer system ofclaim 11 wherein the illumination light source provides light having apower of at least about 250 mW.
 16. The Raman spectrometer system ofclaim, 12 wherein the monochromatic illumination light source provideslight having a wavelength of about 785 nm, and a power of about 300 mW.17. The Raman spectrometer system of claim 12 wherein the proximal endof the detection light guide is optically connected to the probe. 18.The Raman spectrometer system of claim 17 wherein the probe comprises acompound parabolic concentrator optically connected at the proximal endof the detection light guide, wherein the compound parabolicconcentrator collects light emanating from the sample and concentratesit and transmits it into the distal end of detection light guide. 19.The Raman spectrometer system of claim 17 wherein the proximal end ofthe detection light bundle has a diameter greater than about 1 mm andthe detection probe does not comprise a compound parabolic concentrator.20. The Raman spectrometer system of claim 17 wherein the probe is sizedto provide an illumination spot on the sample that is substantiallylarger than a detection spot detected by the probe.
 21. The Ramanspectrometer system of claim 9 or 10 wherein the substantially inverseshape is a parabolic curve.
 22. The Raman spectrometer system of claim 9or 10 wherein the plane grating is a volume phase technology (VPT)holographic grating.
 23. The Raman spectrometer system of claim 9 or 10wherein the light transmissive portion of the proximal end of thedetection light guide comprises a filled-in geometrical shape.
 24. TheRaman spectrometer system of claim 23 wherein the filled-in geometricalshape is a circle.
 25. The Raman spectrometer system of claim 9 whereinthe detection light guide further comprises a calibration light guideoptically connected to a calibration light source.
 26. The Ramanspectrometer system of claim 25 the calibration light guide is disposedat the center of the detection light guide.
 27. The Raman spectrometersystem of claim 9 wherein detector comprises an array of detectionpixels and the detection light guide comprises a bundle comprisingenough light guides to substantially fill a column or row of the array.28. The Raman spectrometer system of claim 27 wherein bundle comprisesmore than about 50 light guides.
 29. The Raman spectrometer system ofclaim 28 wherein the light guides are selected from the group consistingof an optical fiber, a liquid light guide and a hollow reflective lightguide.
 30. The Raman spectrometer system of claim 9 wherein the systemis portable.
 31. A light transmission means suitable for use for Ramanspectroscopy, the light transmission means comprising a means forreceiving light in a substantially filled-in geometrical shape at aproximal end of the light transmission means and means for emitting thelight in a substantially linear curve at a distal end of the lighttransmission means.
 32. The light transmission means of claim 31 whereinthe substantially linear curve is substantially identical to a curve ofa substantially linear line of light after it has been passed through aholographic grating.
 33. The light transmission means of claim 31wherein the light transmission means further comprises a means fortransmitting calibration light.
 34. A Raman spectrometer systemcomprising a means for transmitting detection light emanating from asample, a distal end of the detection means optically connected to aplane grating means that is in turn optically connected to a means fordetecting that is operably connected to a controller means containingcomputer-implemented programming that detects light impinging on meansfor detecting, wherein the means for transmitting detection lightprovides light in a substantially inverse shape that is complementary toa distortion to the light caused by passing the light through the planegrating, to provide light in a substantially straight line at the meansfor detecting.
 35. The Raman spectrometer system of claim 34 wherein thesystem further comprises a means for providing monochromaticillumination light to the sample.
 36. The Raman spectrometer system ofclaim 35 wherein the system further comprises a probe means for emittingillumination light to the sample and for receiving light emanating fromthe sample, the probe means optically connected to a distal end of ameans for transmitting illumination light and to a proximal end of themeans for transmitting detection light.
 37. The Raman spectrometersystem of claim 36 wherein the probe means provides an illumination spoton the sample that is substantially larger than a detection spotdetected by the probe means.
 38. The Raman spectrometer system of claim31 wherein the system further comprises a means for transmittingcalibration light.
 39. The Raman spectrometer system of claim 31 whereinthe system is portable.
 40. A method of making a light transmissionbundle comprising a) providing at least 5 light guides; b) arranging thelight guides in a filled-in geometrical shape at a proximal end of thelight transmission bundle; and c) arranging the light guides in asubstantially linear curve at the distal end of the light transmissionbundle.
 41. The method of claim 40 wherein the light guides are arrangedin a substantially parabolic curve at the distal end of the lighttransmission bundle.
 42. The method of claim 41 wherein the paraboliccurve is substantially identical to a curve of a substantially linearline of light after it has been passed through a holographic grating.43. The method of claim 40 wherein the filled-in geometrical shape is acircle.
 44. The method of claim 40 wherein the method further comprisesadding a calibration light guide to the light transmission bundle.
 45. Amethod of taking a Raman measurement comprising: a) illuminating asample under conditions and for a time sufficient to induce measurableRaman scattered light to emanate from the sample; b) collecting theemanating light; c) providing the emanating light with a substantiallyinverse shape that is complementary to a distortion to the light causedby passing the light through a light-dispersive element; d) passing thelight having the substantially inverse shape through the plane gratingto provide substantially straight spectral lines; and, e) performingRaman spectroscopic analysis on the substantially straight spectrallines.
 46. The method of claim 45 wherein the providing thesubstantially inverse shape further comprises transmitting the emanatinglight through a detection light guide wherein the light transmissiveportion of the distal end of the detection light guide is arranged in asubstantially inverse shape that is complementary to a distortion to thelight caused by passing the light through the light transmissiveelement, and wherein the light transmissive element comprises a planegrating.
 47. The method of claim 45 wherein the method further comprisesilluminating the sample and collecting the emanating light through aprobe optically connected to an illumination light source and to thedetection light guide.
 48. The method of claim 45 wherein the methodfurther comprises providing an illumination spot on the sample that issubstantially larger than the detection spot detected by the probe. 49.The method of claim 45 wherein the method further comprises providingcalibration light.
 50. The method of claim 45 wherein the method isperformed using a portable Raman system.
 51. The method of claim 45wherein the Raman measurement is obtained in vivo.
 52. The method ofclaim 45 or 51 wherein the Raman measurement is obtained in less thanabout 1 second.